Systems and methods for patterning and spatial electrochemical mapping of cells

ABSTRACT

Disclosed herein are an apparatus for electrically assessing and/or manipulating cells. One aspect is directed to electrically mapping cells on the surface of the semiconductor substrate via cross-electrode impedance measurements. Further according to some aspects, the electrode array allows for spatially addressable electrical stimulation and/or recording of electrical signals in real-time using the CMOS circuitry. Some of these aspects are directed to using an electrode array to perform cell patterning through electrochemical gas generation, and extracellular electrochemical mapping.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 63/040,439, filed Jun. 17, 2020, entitled “Systemsand Methods for Patterning and Spatial Electrochemical Mapping ofCells,” by Park, et al. which is incorporated herein by reference in itsentirety.

BACKGROUND

The present disclosure relates to a semiconductor device forelectrically assessing cells or other biological specimens.

SUMMARY OF THE DISCLOSURE

Disclosed herein are various apparatuses for electrically assessingand/or manipulating cells. One aspect is directed to electricallymapping cells on the surface of the semiconductor substrate viacross-electrode impedance measurements. Further according to someaspects, the electrode array allows for spatially addressable electricalstimulation and/or recording of electrical signals in real-time usingthe CMOS circuitry. Some of these aspects are directed to using anelectrode array to perform cell patterning through electrochemical gasgeneration, and extracellular electrochemical mapping.

Some embodiments relate to an apparatus for mapping one or more cells.The apparatus comprises a semiconductor substrate. The semiconductorsubstrate comprises a plurality of electrodes exposed at a surface ofthe semiconductor substrate; active circuitry coupled to the pluralityof electrodes and configured to measure a first set of cross-electrodecurrents between a first electrode of the plurality of electrodes andsome or all of the remaining electrodes; measure a second set ofcross-electrode currents between a second electrode of the plurality ofelectrodes and some or all of the remaining electrodes. The apparatusfurther comprises one or more processors configured to receive themeasured cross-electrode currents from the active circuitry and togenerate a map of the one or more cells based on the first set andsecond set of cross-electrode currents.

In some embodiments, the active circuitry is further configured to applya stimulus signal at the first electrode of the plurality of electrodes,and to apply a reference voltage at the remaining electrodes where thecross-electrode currents are being measured from. The stimulus signalmay have a frequency of less than 10 kHz and preferably between 0.1 and5 kHz. The plurality of electrodes may be arranged in an array having apitch of less than 30 μm and preferably less than 5 μm. Thesemiconductor substrate may comprise silicon. The semiconductorsubstrate may comprise a silicon substrate, and the active circuitry maycomprise complimentary metal-oxide semiconductor (CMOS) components inthe silicon substrate. The plurality of electrodes may comprise aplurality of pads disposed on an insulative surface of the semiconductorsubstrate. The active circuitry may comprise a plurality of recordingcircuits, each recording circuit configured to measure a current at anelectrode of the plurality of electrodes. The plurality of recordingcircuits may comprise at least 8 recording circuits, at least 10recording circuits, and preferably at least 4000 recording circuits.Each recording circuit may comprise a transimpedance amplifier (TIA).The TIA may comprise an impedance component having a resistance of atleast 10 MΩ, at least 100 MΩ, or between 10 MΩ and 1 GΩ, wherein anoutput voltage of the TIA is proportional to a voltage across theimpedance component. The impedance component may comprise a switchingcapacitor. The one or more cells may be disposed in a first well of amulti-well plate, and the plurality of electrodes may be a firstelectrode array exposed to the first well, and the apparatus further maycomprise a second electrode arrays exposed on the surface of thesemiconductor substrate, and exposed to a second well of the multi-wellplate. The multi-well plate may comprise at least 24, at least 96, or atleast 384 wells. The plurality of electrodes may be sized such that morethan one electrode are configured to be in contact with one cell of theone or more cells. The plurality of pads may comprise Au. The pluralityof pads may comprise Pt.

Some embodiments relate to a method for mapping one or more cells incontact with an electrode array disposed on a surface area of asemiconductor substrate. Each electrode in the electrode array has anelectrode location on the surface area. The method comprises for eachelectrode of at least one electrode of the electrode array, applying astimulus signal at the electrode; measuring a set of cross-electrodecurrents between the electrode and some or all of the remainingelectrodes in the electrode array; generating a representative valueassociated with the electrode location of the electrode based on the setof cross-electrode currents; and generating a map of representativevalues on the surface area based on the generated representative valuesand respective associated electrode locations of the at least oneelectrode.

In some embodiments, generating the representative value comprisesselecting a maximum current value of the set of cross-electrode currentas the representative value. Generating the representative value maycomprise selecting a maximum current value of the set of cross-electrodecurrent as the representative value. The at least one electrode mayinclude all electrodes in the electrode array. The map may have aspatial resolution of 20 μm or less and preferably 5 μm or less.

In some embodiments, the generated map is a first map generated at afirst time and comprises a plurality of pixels, and the method furthercomprises: generating a second map of representative values on thesurface area at a second time subsequent to the first time, wherein thesecond map comprises a plurality of pixels; determining a first count ofpixels in the first map having a representative value within apredetermined range; determining a second count of pixels in the secondmap having a representative value within the predetermined range; anddetermining a cell adhesion characteristic based on a comparison of thefirst count with the second count. The map may comprise a plurality ofpixels, each pixel associated with a representative value. The at leastone electrode may comprise a first electrode having a first electrodelocation and a second electrode having a second electrode location, thefirst electrode and the second electrode adjacent each other on thesurface area, and the map may comprise a first pixel and a second pixelcorresponding to the first electrode location and the second electrodelocation, respectively. Generating the map may comprise determining anup-scaled representative value associated with a third pixel between thefirst and second pixels. Determining the up-scaled representative valuemay comprise calculating an up-scaled electrode current by dividing across-electrode current I₁₂ between the first and second electrode whena stimulus signal is applied at the second electrode with a product of afirst current I₁ and a second current I₂, wherein I₁ is a sum ofcross-electrode currents measured at all of the remaining electrodeswhen a stimulus signal is applied at the first electrode, and I₂ is asum of cross-electrode currents measured at all of the remainingelectrodes when a stimulus signal is applied at the second electrode. Anumber of pixels in the map may be more than a number of electrodes inthe electrode array. Electrode locations in the electrode array may bearranged in a plurality of rows and a plurality of columns. Theelectrode array may have M rows and N columns, and the map may have atleast 3M×3N pixels.

Some embodiments relate to a system for mapping one or more cells. Thesystem comprises a plurality of electrodes exposed at a surface area ofa semiconductor substrate; circuitry disposed in the semiconductorsubstrate that is controllable to apply a stimulus signal and/or measurea current at one or more electrodes of the plurality of electrodes; atleast one non-transitory computer-readable medium having stored thereonexecutable instructions; and at least one processor programmed by theexecutable instructions to perform a method. The method comprises actsof: for each electrode in the plurality of electrodes, controlling thecircuitry to apply a stimulus signal at the electrode; controlling thecircuitry to measure a set of cross-electrode currents between theelectrode and some or all of the remaining electrodes in the pluralityof electrodes; generating a representative value associated with theelectrode location of the electrode based on the set of cross-electrodecurrents; and generating a map of representative values on the surfacearea based on the generated representative values and respectiveassociated electrode locations of the plurality of electrodes.

In some embodiments, generating the representative value comprises:selecting a maximum current value of the set of cross-electrode currentas the representative value. Generating the representative value maycomprise selecting a maximum current value of the set of cross-electrodecurrent; and calculating an impedance based on the selected maximumcurrent value as the representative value. The map may comprise aplurality of pixels, each pixel associated with a representative value.The plurality of electrodes may comprise a first electrode having afirst electrode location and a second electrode having a secondelectrode location, the first electrode and the second electrodeadjacent each other on the surface area. The map may comprise a firstpixel and a second pixel corresponding to the first electrode locationand the second electrode location, respectively, and generating the mapmay comprise determining an up-scaled representative value associatedwith a third pixel between the first and second pixels.

Some embodiments relate to a method for providing spatially positionedelectrochemical reactions with an electrode array exposed on a surfaceof a semiconductor substrate. The method comprises selecting one or moreelectrodes in the electrode array; controlling circuitry in thesemiconductor substrate to apply, at the one or more electrodes, one ormore stimulus signals to initiate an electrochemical reaction at the oneor more of electrodes.

In some embodiments, the electrochemical reaction may be a half reactionthat generates a gas in a solution, and the one or more stimulus signalsmay comprise potentials that are above a redox potential for generationof the gas. The solution may comprise a plurality of cells attached tothe surface of the semiconductor substrate, and the method may furthercomprise: generating the gas at the selected one or more electrodes suchthat at least one cell of the plurality of cells that is disposed on theselected one or more electrodes is detached from the surface of thesemiconductor substrate. The gas may comprise H₂, Cl₂, or O₂. Theplurality of cells may be a plurality of cells of a first type, and themethod may further comprise: plating one or more cells of a second typeon the surface of the semiconductor substrate at locations where the atleast one cell of the first type has detached from. In some embodiments,the method may further comprise: mapping a time sequence of regrowth ofthe plurality of cells on the surface at positions where the at leastone cell has detached from; and based on the mapping, determining agrowth rate of the plurality of cells. Controlling circuitry to applyone or more pre-determined potentials may comprise performing cyclicvoltammetry at the selected one or more electrodes, and the method mayfurther comprise: measuring, with the circuitry, a value of anelectrical characteristic at each of some or all remaining electrodes inthe electrode array that are outside the selected one or moreelectrodes; and generating a map of electrical characteristics based onthe result of the measuring. The electrical characteristic may be acharacteristic of an open-circuit potential. The electricalcharacteristic may be a current. The characteristic of the current maybe a maximum extent of a range of a cyclic current.

In some embodiments, controlling circuitry to apply one or morepre-determined potentials may comprise applying a pulsed voltage signalat an electrode of the selected one or more electrodes. During a firstportion within the pulsed voltage signal, the electrode is beingoxidized, and during a second portion of the pulsed voltage signal, anoxide on the electrode is being reduced, and the method may furthercomprise: measuring, with the circuitry, a current signal at theelectrode during the second portion of the pulsed voltage signal; basedon a time rate of change of the current signal, determining an oxygenconcentration at a position of the electrode; and generating a map ofoxygen concentration based on the result of the determining. The one ormore potentials may be relative to a potential of a reference electrode.

Some embodiments relate to a system. The system comprises asemiconductor substrate. The semiconductor substrate comprises anelectrode array including a plurality of individually addressableelectrodes disposed on a surface of the semiconductor substrate; andcircuitry that is controllable by one or more processors to apply, at agroup of electrodes in the electrode array, one or more potentialsrelative to a potential of an electrode in the electrode array or apotential of a reference electrode to initiate an electrochemicalreaction at the group of electrodes.

In some embodiments, the electrode array may comprise a plurality ofpads disposed on an insulative surface of the semiconductor substrate.The plurality of pads may comprise Au or Pt. The reference electrode maybe a Ag/AgCl reference electrode. The electrode array may comprise atleast 1000, at least 4000, or at least 1,000,000 electrodes, and thecircuitry may comprise a plurality of recording circuits, each recordingcircuit configured to measure a current at an electrode of the electrodearray. The plurality of recording circuits may comprise at least 10recording circuits, or at least 4000 recording circuits. Each recordingcircuit may comprise a transimpedance amplifier (TIA). The TIA maycomprise an impedance component having a resistance of at least 10 MΩ,wherein an output voltage of the TIA is proportional to a voltage acrossthe impedance component. The impedance component may comprise aswitching capacitor.

Some embodiments relate to a system for providing spatially positionedelectrochemical reactions. The system comprises an electrode arrayexposed at a surface area of a semiconductor substrate; circuitrydisposed in the semiconductor substrate and coupled to the electrodearray; at least one non-transitory computer-readable medium havingstored thereon executable instructions; and at least one processorprogrammed by the executable instructions to perform a method. Themethod comprises acts of: selecting a pattern of electrodes in theelectrode array; and controlling circuitry to apply, at the pattern ofelectrodes, one or more pre-determined potentials relative to apotential of an electrode in the electrode array or a potential of areference electrode, such that an electrochemical reaction is initiatedat the pattern of electrodes.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects and embodiments will be described with reference to thefollowing figures. It should be appreciated that the figures are notnecessarily drawn to scale. Items appearing in multiple figures areindicated by the same reference number in all the figures in which theyappear. In the drawings:

FIG. 1 a is a schematic side view diagram of a semiconductor substrate,in accordance with some embodiments;

FIG. 1B is a two-dimensional data plot of a simulated voltagedistribution in the apparatus shown in FIG. 1 a;

FIG. 1 c is a data plot of simulated electric field lines correspondingto the example shown in FIG. 1B;

FIG. 2 a is a schematic side view diagram of an apparatus with asemiconductor substrate without the presence of a cell, in accordancewith some embodiments;

FIG. 2 b is a schematic side view diagrams illustrating a scenario wherea cell is disposed over some electrodes of the electrode array in FIG. 2a;

FIG. 2 c is a schematic side view diagrams illustrating scenario where acell is disposed outside the electrode array in FIG. 2 a and in betweensome electrodes;

FIGS. 3 a and 3 b illustrate an example of cell mapping usingdistribution of max current;

FIG. 4A is a schematic diagram illustrating an example of a highresolution up-scaled mapping using cross-electrode currents;

FIG. 4B is a schematic circuit diagram of a cell-circuit model;

FIGS. 5 a and 5 b illustrate an example of up-scaled cross-electrodeimpedance mapping in comparison with a fluorescent microscopy image;

FIGS. 6 a-6 c illustrate an example using cross-electrode impedancemapping to quantify cell adhesion;

FIG. 7 is a series of fluorescent microscope images and normalizedcross-electrode impedance maps;

FIG. 8 a is a normalized impedance histogram of a control measurementwithout the tetracycline added;

FIG. 8 b is a normalized impedance histogram of MDCK cells over 6-7 daysof culture in vitro (DIV);

FIG. 9 shows a series of normalized cross-electrode impedance maps underdifferent frequency stimulus signals;

FIG. 10 a illustrates an example of mapping cells and their adhesionover time via a cross-electrode impedance measurement;

FIG. 10 b illustrates an example of measuring cell-to-cell attachment;

FIG. 11 is a schematic diagram illustrating cell patterning throughelectrochemical gas generation;

FIG. 12 illustrates an example of cell spatial patterning and defining aco-culture;

FIG. 13A-D is a series of diagrams illustrating variations of cellpatterning using an electrode array;

FIG. 14 shows a series of fluorescent microscope images illustrating theprocess of defining a co-culture via patterning and then plating asecond cell type;

FIG. 15 is a series of schematic diagrams illustrating a heterogeneouscell population, elimination of undesired cells using patternedelectrochemical gas generation on select electrodes, and a homogenousculture of desired properties after subsequent cell growth;

FIG. 16 illustrates an example of wound healing assay;

FIG. 17 a-d illustrates an experiment demonstrating permeabilizationtechniques;

FIG. 18A-B illustrates an experiment using electroporation protocols, inwhich Fluo-4 is injected into the cell using Fluo-4 AM;

FIG. 19 shows a series of schematic diagrams illustrating generation ofa control and cross-effect delivery using spatial addressing and serialdelivery via gas generation;

FIG. 20 a-b illustrates an example of extracellular electrochemicalmapping;

FIG. 21 a illustrates data plots that show select electrode voltagesplotted over time;

FIG. 21 b-c is a heat map that illustrates for one cycle, the overallamplitude of the open circuit potential plotted across the array;

FIG. 22 a-b illustrates an example of electrochemical oxygen mapping ofcells;

FIG. 23 a shows a series of schematic diagrams illustrating electricalimaging of three parameters useful for live-cell assessment;

FIG. 23 b is an image illustrating a fluidic well packaged on top of achip mounted below a microscope for simultaneous optical and electricalmeasurements;

FIG. 23 c is a colorized microscope image illustrating cells and anelectrode array;

FIG. 23 d is a schematic diagram illustrating an electrode connected toan exemplary pixel circuit;

FIG. 24 a, 24 b are schematic diagrams illustrating some additionalschemes of cell-cell connectivity measurements, in accordance with someembodiments;

FIG. 25 a is a schematic diagram illustrating a pixel amplifierconfigured as a buffer for metabolic state measurement;

FIG. 25 b are a series of data maps showing results of multi-parametricmeasurements;

FIG. 25 c is a pair of nuclei fluorescence images at +72 hours afterplating (top) and a detail region 1 comparison (bottom) showing thelowest cell density on the leading edge in comparison to the trailingedge;

FIG. 25 d is a composite map showing a detail region 2 overlaying thecell nuclei and cell attachment;

FIG. 26 a is a series of fluorescent images illustrating results of acomparison study of electrode impedance under three scenarios;

FIG. 26 b is a data plot illustrating that PtB lowered the Z_(te)measurement of bare electrodes;

FIG. 26 c illustrates cell barrier maps versus a reference at differentfrequencies;

FIG. 26 d shows cell density and connectivity maps extracted from thenuclei of the fluorescence images;

FIG. 26 e shows a comparison between Z_(te) measured without and with areference;

FIG. 26 f shows a comparison between Z_(te) and Z_(s) versus extractedcell density.

DETAILED DESCRIPTION

The present disclosure is directed to various apparatuses forelectrically assessing and/or manipulating cells. In one embodiment, theapparatus includes a semiconductor substrate having complimentarymetal-oxide semiconductor (CMOS) circuitry electrically interfaced withan electrode array that can also be fabricated using CMOS-compatiblefabrication techniques on a surface of the semiconductor substrate andexposed to the cells. The inventors have recognized and appreciated thatby using semiconductor processing techniques, an electrode array may befabricated and integrated with active circuitry in an economicalfashion. Furthermore, electrodes in an electrode array having a smallelectrode size and electrode-to-electrode pitch may allow for higherspatial-resolution assessment of multiple cells compared to using anelectrode that is larger than a size of a cell. For example, individualcells may be distinguishable when mapped using a high density electrodearray, compared to a large electrode covered by an ensemble of cells.Further according to some aspects, the electrode array allows forspatially addressable electrical stimulation and/or recording ofelectrical signals in real-time using the CMOS circuitry. Some of theseaspects are directed to using an electrode array to perform cellpatterning through electrochemical gas generation, and extracellularelectrochemical mapping.

One aspect is directed to electrically mapping cells on the surface ofthe semiconductor substrate via cross-electrode impedance measurements.The inventors have recognized and appreciated that electrical impedancemeasured between two electrodes, or cross-electrode impedance, may beaffected by impedance along a current path between the electrodes. As aresult presence of one or more cells along the current path may affectthe cross-electrode impedance, such that cells can be mapped usingcross-electrode impedance measurements.

FIG. 1 a is a schematic side view diagram of a semiconductor substrate,in accordance with some embodiments. FIG. 1 a shows an apparatus 100that has an electrode array 106 that includes a plurality of electrodes106_1, 106_2, 106_3 . . . 106_n disposed on a surface 104 of asemiconductor substrate 102. FIG. 1 a illustrates an example ofcross-electrode impedance measurement by applying a voltage stimulus toa first electrode such as 106_1, and measuring a current at a secondelectrode such as 106_2. The measured current, also referred to as across-electrode current between electrodes 106_1 and 106_2 flows alongone or more current paths 109 in a medium 108 that is in contact withthe electrode array 106. Electrode 106_1 may be connected to a stimulussource circuit 110, and may be referred to as a stimulation electrode.Electrode 106_2 may be connected to a current measuring circuit 112, andmay be referred to as a recording electrode.

Cross-electrode impedance between electrodes 106_1 and 106_2 may beobtained from the values of cross-electrode current and stimulus voltagebetween the pair of electrodes using any suitable method known in theart, for example by dividing the stimulus voltage amplitude with thecross-electrode current amplitude. A processing unit 120 may be providedthat receives signals from active circuitry within the semiconductorsubstrate 102 and performs the determination of the cross-electrodeimpedance. It should be appreciated that there is no requirement tocalculate the actual impedance values, and that any representativemeasurement that is indicative of impedance between two electrodes maybe used. Alternatively or in addition to calculating the impedancevalue, the cross-electrode current may be used as an indicator for thecross-electrode impedance when measurements at different electrodes arecompared, if the stimulus voltage amplitude is programmed to be a knownconstant.

FIG. 1B is a two-dimensional data plot of a simulated voltagedistribution in the apparatus shown in FIG. 1 a , and shows that when avoltage is applied to a stimulation electrode 106_1, the potential inthe medium 108 falls off both along the vertical direction (V) andlateral direction (L) away from the stimulation electrode 106_1. FIG. 1c is a data plot of simulated electric field lines corresponding to theexample shown in FIG. 1B. FIG. 1 c shows that electric field lines 114that emanate from stimulation electrode 106_1 flows along a line that isdirected upward from electrode 106_1, curves laterally toward recordingelectrodes such as the recording electrode 106_2, before directeddownward to terminate at the recording electrode 106_2.

The presence of cells may alter the shape and distribution of electricfield lines 114 between electrodes and in turn lead to a change incross-electrode impedance, as discussed in detail below in relation toFIG. 2 . FIG. 2 a is a schematic side view diagram of an apparatus 200with a semiconductor substrate 202 without the presence of a cell, inaccordance with some embodiments. In FIG. 2 a , electrode 206_0 of anelectrode array 206 is configured to be a stimulus electrode, withelectric fields lines 214_1 and 214_2 linking stimulus electrode 206_0and recording electrode 206_1. FIG. 2 b is a schematic side viewdiagrams illustrating a scenario where a cell 220 is disposed over someelectrodes of the electrode array in FIG. 2 a . FIG. 2 c is a schematicside view diagrams illustrating scenario where a cell 230 is disposedoutside the electrode array in FIG. 2 a and in between some electrodes.

The inventors have recognized and appreciated that biological cells havea lipid bilayer that forms a continuous membrane barrier around thecell. Electrically, the membrane can behave as a capacitor in parallelwith a high resistance, and can have a different electrical impedancecompared to the surrounding medium such as a solution containing thecells. A cell with its high-impedance membrane on top of the electrodearray will then affect the current distribution in the solution such assolution 208 in FIGS. 2 a-2 c . In FIG. 2 c , a suspended cell blocksfield lines in the solution and lowers the nearest neighbor couplingbetween electrodes. In contrast, a cell attached to the surface andcovering both a stimulation and recording electrode will increase thecross-electrode coupling by blocking vertical field lines.

As an example for the effect of cells on cross-electrode impedance, andwithout wishing to be bound by a particular theory, the inventorsrecognized that if a cell such as cell 220 as shown in FIG. 2 b isadhered to the surface 204 covering some or an entirety of a stimulationelectrode 206_0 and a recording electrode 206_1, the cell 220 willincrease the cross-electrode coupling by blocking electric field lines214_1 and 214_2 between the two electrodes from running verticallythrough the solution 208. As a result, the more vertical field line214_2 is suppressed and the more field line 214_1′ is strengthenedcompared to in FIG. 2 a where no cell is present, resulting in a lowerimpedance between electrodes 206_0 and 206_1.

On the other hand, if a cell is not adhered to the surface 204, or if acell such as cell 230 as shown in FIG. 2 c is adhered to the surface 204but disposed outside of and laterally in between the pair of electrodes206_0, 206_2, the cell may bock electric field lines 214_3 between thepair of electrodes and decrease cross-electrode coupling betweenelectrodes 206_0, 206_2. As a result, cross-electrode impedance mayincrease between electrodes 206_0, 206_2.

Therefore, the presence of a cell above the electrode array and whetherit is adhered to the surface may be detected using cross-electrodeimpedance measurements. It should be appreciated that a cell that isadhered with a surface may have various degrees of non-zero separationbetween the outer extent of the cell membrane and the surface. Anapparatus according to some aspects of the present application mayprovide detection for the degree in which the cell is adhered. Forexample, stronger adhesion will more strongly increase thecross-electrode coupling due to the smaller gap distance along thevertical direction between the cell and the surface of the semiconductorsubstrate.

The cross-electrode measurements may provide several advantages. Forexample, such measurements are non-invasive and can be made repeatedlywithout affecting the cell viability or the electrodes.

In some embodiments and as described above in relation with FIG. 2 b ,the cross-electrode impedance technique measures an increase incross-electrode coupling between electrode pairs due to suppression ofvertical electric field lines from the presence of a cell, as opposed totechniques that measure a decrease in cross-electrode coupling (or anincrease in the measured impedance) due to blocking from the presence ofa cell. One advantage for using the increase in cross-electrode couplingas indicator to detect cell presence is that the increase is mainlyattributed to electrode pairs that are close to each other, in somecases to nearest neighbor coupling between electrode pairs. Thereforethe increase in cross-electrode coupling (or decrease in the measuredimpedance) can be separated from the total background current flowingthrough the stimulation electrode to the many remaining electrodes inthe electrode array. As a result, signal-to-background ratio andsensitivity of the cell detection can be improved.

In contrast to the cross-electrode impedance technique, the inventorshave recognized that simple impedance measured at individual electrodeswould fail to detect the presence of cells. In such a measurement on oneelectrode, the sum of all the return current is measured as the signalfor the impedance on the electrode. Namely, such a measurement is animpedance measurement of an electrode only, and are not measuring thechange of the electric field in the solution on the electrodes. As aresult, the inventors have observed that the impedance of the electrodeitself is not sensitive to the presence of a cell even if the cell isculture directly on its surface.

Referring back to FIG. 1 a , in some embodiments, a stimulus signalapplied by stimulus source circuit 110 to the stimulus electrode 106_1is a low frequency alternative current (AC) signal, having a frequencyof less than 10 kHz, less than 5 kHz, between 0.1 and 5 kHz, or between0.1 and 2 kHz. The low frequency stimulus signal is selected because thecell membrane acts as a capacitor in parallel with a high resistance,and at high frequency the capacitor impedance would decrease and renderthe cell highly conductive. The inventors have recognized andappreciated that by measuring cross-electrode current at low frequencycan provide high signal contrast for detection of cell adhesion. Anexample of the frequency response of cross-electrode impedancemeasurement is provided in Example 4 below.

Still referring to FIG. 1 , the semiconductor substrate 102 may includean active circuitry 116. Active circuitry 116 may include a plurality ofstimulation circuits 110 and a plurality of recording circuits 112. Insome embodiments, the stimulation circuit 110 may comprise one or morecurrent injectors, one or more voltage sources, or a combinationthereof. Some aspects of the active circuitry design are related tocurrent-based stimulators for electrogenic cells and related methods, asdisclosed in International Application Publication. No. WO 2019/010343,Attorney Docket No. H0776.70105WO00, the disclosure of which is herebyincorporated by reference in its entirety. Some aspects may also berelated to electronic circuits for analyzing electrogenic cells andrelated methods, as disclosed in International Application Publication.No. WO 2019/089495, Attorney Docket No. H0498.70647WO00, the disclosureof which is hereby incorporated by reference in its entirety. In someembodiments, the active circuitry may comprise programmable currentinjectors for performing current-voltage measurements using one or moreof the electrodes in the electrode array as working and/or counterelectrodes.

In some embodiments, each recording circuit comprises a transimpedanceamplifier (TIA) having a switching capacitor as impedance component. Theresistance of the impedance component is at least 10 MΩ (megohms), atleast 100 MΩ (megohms), or between 10 MΩ and 1 GΩ (gigohm) to provideamplification of a recorded current signal at an input of the TIA,whereas an output of the TIA provides an output voltage that isproportional to the recorded current signal and to a voltage across theimpedance component.

Electrodes in the electrode array 106 may be reconfigured using theactive circuitry 116 as a stimulation electrode or as a recordingelectrode. In some embodiments, active circuitry 116 comprises routingand switching components that are programmable to connect a selectedelectrode of the electrode array 106 to stimulus source circuit 110, tocurrent measuring circuit 112, or to other circuit components to enabledifferent functionalities. Depending on the application, more than oneelectrode may be configured as a stimulus electrode, and more than oneelectrode may be recording at the same time. For example, when mappinglocal cell properties using cross-electrode impedance measurements,typically only one electrode acts as stimulus electrode at a time. Insome other embodiments, a subset of one or more electrodes may beselected to act as a stimulus or to apply one or more potentials orcurrents to initiate an electrochemical reaction at the locations of theselected one or more electrodes. The latter embodiments will bediscussed in more detail in the sections below regarding cell-to-cellattachment measurement, patterning, and spatial electrochemical mappingof cells.

In some embodiments, the electrodes may be biased using low impedancesources/returns in the active circuitry. For example, a lowoutput-impedance voltage source may be used to provide a stimulus signalat a stimulus electrode, while a low input-impedance transimpedanceamplifier may be provided for current measurement at a recordingelectrode. In such embodiments, each electrode may be selectivelyconnected to a voltage source for stimulation, to a transimpedanceamplifier for current measurement, to a voltage source for a return, orto a transimpedance amplifier for simultaneous stimulation and currentmeasurement. The inventors have appreciated and recognized that the lowimpedance source/return may facilitate formation of fringing electricfield lines in the solution as illustrated in the example in FIG. 1 c.

Semiconductor substrate 102 may comprise silicon, and in suchembodiments, active circuitry 116 may be an integrated circuit thatcomprises CMOS components fabricated using standard CMOS processingtechniques. The electrode array 106 may be disposed within semiconductorsubstrate 102, for example as conductors exposed from a surface 104 ofthe semiconductor substrate 102 that faces the medium 108. In someembodiments, the surface 104 is an insulative surface that providesmechanical support and electrical isolation to the electrode array 106while also providing a suitable surface for cells to grow. While FIG. 1a shows that the electrode array 106 is partially embedded in thesemiconductor substrate 102, such an arrangement is an illustrativeexample only and not a requirement. In some embodiments, the topsurfaces of electrodes in electrode array 106 may be above, alignedvertically with, or below the surface 104 of the semiconductor substrate102. Additionally or alternatively, the top surfaces of the electrodesmay have a passivation layer or functionalization layer. In someembodiments, holes may be patterned in the passivation orfunctionalization layer on top of the electrodes to expose theconductive surfaces of the electrodes to the medium.

It should be appreciated that semiconductor substrate 102 may be anysubstrate fabricated using semiconductor processing techniques, and notlimited to a silicon wafer. For example, semiconductor substrate 102 maycomprise group IV semiconductor, III-V semiconductor, II-Vsemiconductor, sp² hybridized carbon material, chalcogenide, metal,metallic compound, oxide, nitride, silicide, polymer material, orcombinations thereof. Semiconductor substrate 102 may be a unitarycomponent, or a composite of multiple components. Components in thesemiconductor substrate 102 may comprise an active circuit layer, awiring layer, a redistribution layer, a circuit board, or combinationsthereof. Component layers in the semiconductor substrate may be formedin the addition process during CMOS processing, or be formed separatelyand bonded to each other using packaging techniques known in the field.Conductors are provided in the semiconductor substrate 102 thatinterconnects active circuitry 116 with the electrode array 106. In someembodiments, connection points are provided at a bottom surface of thesemiconductor substrate for electrically interfacing components withinthe semiconductor substrate with processing unit 120. Electricalconnection between processing unit 120 and the semiconductor substrate102 may be provided via any suitable way, such as but not limited tocontrolled collapse chip connection or flip chip bonding, wire bonding,flexible cables, or wireless communication.

Referring back to FIG. 1 a . In some embodiments, apparatus 100 may beoperated to perform a method, such as mapping or performing selectiveelectrochemistry. The operation of the apparatus 100 may be underprogram control. In some embodiments, processing unit 120 in apparatus100 may comprise a computer 20 with storage media 21, memory 23, andprocessor 25, and such processing may be performed in computer 20 or anyother computing device. Storage media 21 and memory 23 may be anysuitable non-transitory computer-readable medium, such as, for exampleand not limited to a computer memory, compact discs, optical discs,magnetic tapes, flash memories, circuit configurations in FieldProgrammable Gate Arrays (FPGAs) or other semiconductor devices, orother tangible computer storage medium. In some embodiments, storagemedia 21 may be non-volatile storage and memory 23 may be volatilestorage. Computer-executable instructions may be loaded from storagemedia 21 to memory 23 before execution by processor 25 to perform someor all of the methods as described throughout the present disclosure.However, a distinction between storage media 21 and memory 23 is notcritical and either or both may be present in some embodiments.

Processor 25 may be any suitable processing device, such as, for exampleand not limited to, one or more processors, a central processing unit(CPU), digital signal processor (DSP), controller, addressablecontroller, general or special purpose microprocessor, microcontroller,addressable microprocessor, programmable processor, programmablecontroller, dedicated processor, dedicated controller, or any othersuitable processing device. Some or all components within processingunit 120 may be packaged as a system-on-a-chip (SOC). Moreover, itshould be appreciated that FIG. 1 a is a schematic representation of aprocessing unit 120. An actual implementation of a processing unit 120may have distributed processing. A host computer, for example, maycontrol the overall flow of measurement, mapping and analysis ofresults.

Turning now to the electrode array 106. In some embodiments, electrodearray 106 may be patterned on the surface 104 as part of thesemiconductor fabrication process to form the active circuitry 116within semiconductor substrate 102, and may be conductive pads thatcomprise metal such as Au or Pt, or alloys thereof. For example, thepads may be formed of Al with plated Au as a top layer. In suchembodiments, substrate 110 may additionally comprise conductors thatinterconnect vertically the exposed electrode array 14 to circuitrywithin substrate 110.

Electrodes in the electrode array 106 may be arranged on the surface 104in any suitable arrangement, such as a two-dimensional array withregular pitches along the row and column directions. In some embodimentsof the cross-electrode impedance-based mapping, a pitch of the electrodearray may be selected to be on the order of or smaller than a size oftypical cells such that a cell can cover at least two electrodes toincrease coupling between the cell and the at least two electrodes. Forexample, when the size of cells is about 30 μm, the pitch of theelectrode array may be set as less than 30 μm, less than 20 μm, lessthan 5 μm, or between 1 and 20 μm. Providing a small pitch betweenelectrodes allows a cell to cover two or more electrodes, which permitsmeasuring the cell-to-substrate gap distance via an increase incross-electrode coupling at the electrodes under the cell.

In some embodiments where the electrode array is fabricated during aCMOS-compatible fabrication process on top of the semiconductorsubstrate containing CMOS active circuitry, the pitch of the electrodearray and size of each electrode may be selected by taking intoconsideration the pitch and density of the CMOS active circuitry. Forexample, in some embodiments at least 8, at least 10, or at least 4000recording circuits may be provided within the semiconductor substrate,and the electrode array may have at least 1000, or at least 4000, or atleast 1,000,000 electrodes. In such embodiments, each electrode may havea lateral dimension of no more than 10 μm, or no more than 5 μm, suchthat the overall lateral extent of the electrode array is containedwithin the surface of the semiconductor substrate. An electrode arrayaccording to aspects of the present disclosure may also be referred toas a CMOS microelectrode array (MEA).

Referring back to FIG. 1 , the medium 108 may be a cell culture medium,and may be a solution that comprises any number of chemical and/orbiological reagents in addition to cells. While not shown in FIG. 1 ,medium 108 may be contained in a container disposed on top of thesemiconductor substrate 102. In some embodiments, the container may be awell of a multiple-well plate attached to the semiconductor substrate,with one or more wells having an open bottom exposing contents of thewell to the semiconductor substrate. The semiconductor substrate maycomprise more than one electrode arrays, such that electric assessmentin multiple wells may be conducted in parallel.

CMOS-compatible, wafer-scale, multi-well platform that can be used forbiomedical or other applications, and methods to operate the same. Insome applications, circuitry is provided underneath a multiple-wellarray to electrically interface with electrodes in the wells. Theplatform may sometimes be referred to as a CMOS-Multiwell Platform. Theinventors have recognized and appreciated that to interface withelectrodes in a large array, circuitry may be fabricated on a singlesilicon (Si) wafer having a dimension that is at least the same orlarger than that of the multiple-well array. According to one aspect ofthe present disclosure, standard CMOS fabrication processes such asthose known to be used in a standard semiconductor foundry may be used,e.g., without expensive customization for complex fabricationprocedures, and thus the production cost can be lowered in some cases.The CMOS-Multiwell Platform according to some aspects of this disclosurecan be used in applications including electrophysiology studies andgeneral cell assessment using electrical methods, and/or in a highthroughput format (e.g. 24-, 96-, and 384-well plate formats).

In some embodiments, the Si wafer is part of a semiconductor device, andhas an array of reticle areas, with some or all of the reticle areashaving a plurality of circuitry of a same design. The inventors haverecognized and appreciated that during manufacturing, reticle areas of awafer may reuse the same lithographical mask design repeated across thewafer in some cases, thus reducing the cost of tooling and increasingthe wafer manufacturing throughput.

According to an aspect, digital and analog circuitry within a reticlearea may be arranged to correspond to one or more wells when themultiple-well array is coupled on top of the wafer. Some embodiments cantherefore provide a wafer-scale integration of electrical interface witha multiple-well array by using a manufacturing method that does not dicethe wafer and/or is compatible with standard using standardCMOS-compatible techniques to reduce manufacturing cost.

One aspect of the present disclosure is directed to a technique ofmapping the spatial distribution and dimensions of cells usingcross-electrode impedance measurements. The mapping may additionallyrepresent a property of individual cells such as adhesion to the surfaceof a semiconductor substrate. In some embodiments, because cell presenceis primarily reflected locally in cross-electrode coupling between astimulus electrode and nearby recording electrodes, mapping is performedby first choosing an individual electrode as stimulus electrode, andmeasuring a set of cross-electrode impedance data against otherelectrodes at locations throughout the electrode array. Subsequently, adifferent electrode is chosen as stimulus electrode, and a new set ofcross-electrode impedance data is measured. The cross-electrodemeasurements are repeated by sequentially setting electrodes in theelectrode array to apply a stimulus signal, and the corresponding set ofmeasurement cross-electrode impedance data may then be processed togenerate a value that indicates for each location of the stimuluselectrode, whether there is a presence of a cell, or a strength of acell property. The processed values may then be combined to form a mapacross the area of the electrode array. In some embodiments,“electro-chemical imaging” of live-cell cultures are demonstrated byhigh-resolution in situ impedance and electrochemical measurement. Someembodiments are directed to using CMOS-MEAs to perform label-free andnon-invasive tracking of cell growth dynamics and accurate measurementsof cell-substrate attachment, cell-cell adhesion, and metabolic state.

Another aspect is directed to providing spatially positionedelectrochemical reactions using a patterned electrode array. With aselected number of electrodes in the electrode array, active circuitryin the semiconductor substrate may apply potentials to initiate anelectrochemical reaction in the solution regions directly above theselected electrodes. As a result, electrochemistry can be performedselectively at a programmed spatial pattern, based on the size, shapeand distribution of the selected electrodes on the surface of thesemiconductor substrate.

In some embodiments, spatially programmed electrochemistry may be usedto perform cell patterning. For example, cells adhered to an electrodemay be selectively removed from the electrode surface byelectrochemically generate small gas bubbles on the electrode.

In some embodiments, an array of electrochemical electrodes may be usedto spatially map analyte concentrations as measured using activecircuitry in the semiconductor substrate. One application is anelectrochemical mapping of solutions using redox electrochemistry.

The following applications are each incorporated herein by references intheir entireties: U.S. Provisional Patent Application Ser. No.63/040,439, filed Jun. 17, 2020, by Park, et al.; U.S. ProvisionalPatent Application Ser. No. 63/040,424, filed Jun. 17, 2020, by Ham, etal.; and U.S. Provisional Patent Application Ser. No. 63/040,412, filedJun. 17, 2020, by Ham, et al. In addition, the following are eachincorporated herein by references in their entireties: a PCT patentapplication, filed on Jun. 16, 2021, entitled “ComplementaryMetal-Oxide-Semiconductor (CMOS) Multi-Well Apparatus for ElectricalCell Assessment” and a PCT patent application, filed on Jun. 16, 2021,entitled “Apparatuses for Cell Mapping Via Impedance Measurements andMethods to Operate the Same.”

The following examples are intended to illustrate certain embodiments ofthe present invention, but do not exemplify the full scope of theinvention.

Example 1: Real-Time Cell Measurements Using a CMOS Microelectrode Array(MEA) and Imaging System

This example describes electrical imaging of three parameters useful forlive-cell assessment (FIG. 23 a ): cell-substrate impedance, Zs(reflecting cell attachment and cell-substrate adhesion),transepithelial impedance, Zte, (reflecting cell-cell adhesion and theintegrity and barrier function of the cell monolayer), and extracellularredox potential, Vredox (reflective of the cellular metabolic state andrespiration).

In this example, a custom designed CMOS IC is used that parallelizesimpedance and electrochemical capabilities across a 64×64=4,096 array ofelectrodes (FIG. 23 b-d ). A fluidic well is packaged on top of the chipto culture cells and is mounted below a top-down fluorescence microscopefor simultaneous optical and electrical measurements (FIG. 23 b ). Thearray of electrodes sits at the center of the device, consisting of 8 μmdiameter Pt electrodes spaced at a 20 μm pitch for single- or few-cellresolution (e.g. MDCK cells in FIG. 23 c ), and results in a totalsensing area of 1.26×1.26 mm². The remainder of the surface is insulatedwith silicon nitride which behaviors similar to glass culture plates. Nodifference is observed in growth or morphology for cells cultured on thedevices in comparison to traditional culture plates. For long-termmeasurements, an integrated temperature sensor and heater regulate thecells to 35-37° C. and a mini-incubation chamber is placed over thedevice to regulate CO₂ to 5%.

Each electrode in the array is connected to its own pixel circuit (FIG.23 d ) which is highly configurable and programmed via a digitalinterface. The pixel circuit comprises an operational amplifier whichcan be configured as a buffer for electrode voltage, Ve, measurement, oras a transimpedance amplifier for electrode current, Ie, measurement.Some aspects of the pixel circuit configuration are related tocurrent-based stimulators for electrogenic cells and related methods, asdisclosed in International Application Publication. No. WO 2019/010343,Attorney Docket No. H0776.70105WO00, the disclosure of which is herebyincorporated by reference in its entirety. Some aspects may also berelated to electronic circuits for analyzing electrogenic cells andrelated methods, as disclosed in International Application Publication.No. WO 2019/089495, Attorney Docket No. H0498.70647WO00, the disclosureof which is hereby incorporated by reference in its entirety.

FIG. 23 a are schematic diagrams that illustrate three cell parametersthat are electrically measured using a complementarymetal-oxide-semiconductor (CMOS) integrated circuit (IC) for live-cellassessment: cell attachment via a cell-substrate impedance, Z_(s),cell-cell adhesion via a transepithelial impedance, Z_(te), and themetabolic state via the extracellular redox potential, Vredox. Eachmeasurement is non-invasive and fast (<1 min), allowing the measurementsto be repeated sequentially every 5-10 min for real-time investigations.FIG. 23 b is a picture showing that a fluorescent microscope can bepaired with the packaged CMOS IC for simultaneous optical and electricalcell measurement. A reference electrode, Pt (shown) or Ag/AgCl, can bealso be used in this example. FIG. 23 c is a colorized fluorescent imageof Madin-Darby Canine Kidney (MDCK) epithelial cells cultured on top ofthe CMOS electrode array. The 64×64=4,096 circular 8 μm diameterplatinum electrodes are spaced at a 20 μm pitch. Platinum black (PtB)can be electrodeposited onto the electrodes to lower the electrodeimpedance for higher signal-to-noise Z_(te) measurement. FIG. 23 d is acircuit diagram of an exemplary circuit for an electrode in theelectrode array. Each of the 4,096 electrodes is connected its ownperipheral circuit via a shielded routing (˜1-10 mm). The op-amp basedcircuit can be configured to apply a voltage via V_(s) and measure acurrent via a feedback resistor R_(f) (˜100 MΩ), or to apply a currentvia I_(s) and buffer/measure the electrode voltage, Ve. The output ofthe op-amp, V_(amp), is routed off-chip for analog-to-digitalconversion. The switches are digitally programed using a real-timesoftware interface.

In accordance with some aspects, the high channel count (4,096),parallel current and open-circuit potential measurements featured in themeasurement techniques in this example provide unique advantages overother MEA devices. For example, measurements as described in thisexample are prevented in MEA devices that measure the electrodecapacitance, voltage with high-pass filters to block DC signals, orcurrent with a small number of channels (<32).

Example 1A: Cell Mapping Using Distribution of Max Current

This example describes a technique of mapping cells using a CMOSelectrode array which contains a 64×64 array of 4,096 platinumelectrodes at a 20 μm pitch.

The inventors have recognized and appreciated that alternating current(AC) impedance measurements between a pair of electrodes can detectcells using the contrast between the insulative cell membrane andconductive culture media. In a classic impedance measurement, solutionpaths around the cells shunt the measurement and lower detectionsensitivity, as the solution contribution of the measuredelectrode-to-electrode current is far larger than the small change ofcurrent due to the cells. The device as disclosed herein improvesdetection sensitivity by instead measuring a change of electric fielddistribution due to the cells.

An AC voltage (1.9 kHz frequency, 200 mV amplitude) was applied to oneelectrode and the resultant AC currents were measured through theremaining 4,095 electrodes using transimpedance amplifiers. The resultis illustrated in FIG. 3 a , which shows a measured current distributionheat map 301 of the nearest 11×11 recording electrodes to the onestimulus electrode 311 when no cell is present. In heat map 301, eachpixel corresponds to a location of an electrode. Each electrode has anelectrode location or electrode position that may be expressed in anumber of ways, such as but not limited to a coordinate or a pixelnumber. Heat map 302 is a measured current distribution that is similarto heat map 301, but with a cell on top of the electrode 311. Theimpedance measurements were done with a 1.9 kHz signal frequency.

The measured cross-electrode current versus distance to stimulationpixel data plot 303 in FIG. 3 a shows that in the presence of a cell,the cross-electrode coupling to adjacent electrodes is higher by almostan order of magnitude in comparison to electrodes without cells on top.

In this example, a fluorescent nuclei MDCK cell line was used foroptical confirmation. FIG. 3 b shows a fluorescent microscopy image 304across the entire 64×64 electrode array, where the lighter pixelrepresents fluorescent signals that indicate presence of cells. Togenerate a cross-electrode impedance map of the same area as image 304,the stimulation electrode was sequentially scanned across the array. Foreach given stimulation electrode, cross-electrode current values aremeasured from the remaining electrodes as recording electrodes. Therecorded cross-electrode currents are collected and a maximum value isdetermined, referred to as a max current value corresponding to thegiven stimulation electrode. FIG. 3 b shows a heat map 305 across theelectrode array generated using the max current value (Ie) determinedfrom stimulation electrodes at each pixel location.

FIG. 3 b also shows a map 306 that is an overlay of a select region 1 ofthe nuclei fluorescence signals 307 and the max current signals 309showing the ability to map the cluster of cells with single-cellresolution. As a result, this example demonstrates that the presence ofcells was confirmed using nuclei fluorescent markers with a strongcorrespondence between the max current map and fluorescent imaging.

The max current value (Ie) determined for each stimulation electrodelocation using any suitable method based on the set of cross-electrodecurrents measured from the recording electrodes. The determination maybe a simple comparison of absolute arithmetic values of thecross-electrode currents, and may additionally include data processingsuch as noise filtering, background subtraction, or any suitable signalprocessing technique known in the field prior to the comparison.Processing and comparison of the current values may be performed afterdigitization of the measured current values, and using a processing unitsuch as processing unit 120 as shown in FIG. 1 a.

Example 2: High Spatial Resolution Mapping Using Cross-ElectrodeCurrents

This example describes a method to generate an up-scaled map of thecross-electrode coupling that has a higher spatial resolution than thepitch of the electrode array.

According to some embodiments, the nearest neighbor cross-electrodemeasurements may be used for each stimulation electrode. FIG. 4A showsan example of a high resolution up-scaled mapping using a 3×3 impedancegrid for each of electrodes 1-9. In some embodiments, electrodes at theedges of the electrode array may be skipped from the up-scaled impedancegrid as described below.

FIG. 4B is a schematic circuit model that may be used to calculate thecell-substrate impedance, Z_(s), and transepithelial impedance, Z_(te),for the application of an AC stimulation voltage, V_(A), and ameasurement of cross electrode current, I₁₂. The 3×3 impedance grid isused for the Z_(s) calculation while a single Z_(te) is extracted foreach electrode.

To measure the cell-substrate attachment, a change of cross-electrodefield is formed. Instead of applying bias between two electrodes, biasis applied from one electrode to all remaining electrodes. This allowsthe field lines starting from the stimulation electrode and extendingfar up into the culture well to terminate on electrodes far away thestimulation. Otherwise, these field lines would need to curl backtowards the adjacent electrode, increasing the amount of measuredcurrent not related to the immediate cell-electrode interface.

The interface may be modeled using a cross-sectional type model toincrease spatial resolution. If we assume Z_(s)<<Z_(te), Z_(e,1), andZ_(e,2,) which according to some aspects are found to be valid for mostmeasurement, then:

$\begin{matrix}{V_{2} \approx {\frac{1}{2}V_{1}} \approx {\frac{1}{2}V_{A}\frac{2Z_{s}}{Z_{1} + {2Z_{s}}}} \approx {V_{A}\frac{Z_{s}}{Z_{1}}}} & \left( {{{Eq}.A}1} \right)\end{matrix}$

The measured cross electrode current can also be written and expressedin terms of (Eq. A1),

$\begin{matrix}{I_{12} = {\frac{V_{2}}{Z_{2}} \approx {V_{A}\frac{Z_{s}}{Z_{1}Z_{2}}}}} & \left( {{{Eq}.A}2} \right)\end{matrix}$

To determine Z_(e,1) and Z_(e,2), the sum of the measured current acrossthe array is used when the stimulus is applied to an electrode n,

$\begin{matrix}{Z_{e,n} = \frac{V_{A}}{I_{n}}} & \left( {{{Eq}.A}3} \right)\end{matrix}$

Z_(s) can then be solved for from (A3) and (A2),

$\begin{matrix}{Z_{s} = {V_{A}\frac{I_{12}}{I_{1}I_{2}}}} & \left( {{{Eq}.A}4} \right)\end{matrix}$

which uses all measured currents.

To generate a high-spatial map of the Z_(s), nearest neighborcross-electrode measurements were used for each stimulation electrode: a3×3 grid is used for each electrode (except those at the edges of theelectrode array). See FIG. 4A. This creates an overall Z_(s) image of190×190 pixels (in comparison to the 64×64 electrodes in the array).

In the example shown in FIG. 4A, each of the 9 pixels in the 3×3 grid405 for the center electrode 5 is filled in using normalized impedancevalues Z based on the measured currents to its nearest neighboringelectrodes. Each normalized impedance value Z is calculated as,

$\begin{matrix}{{Z_{52} = {V_{AC}\frac{I_{52}}{I_{5}I_{2}}}},{Z_{54} = {V_{AC}\frac{I_{54}}{I_{5}I_{4}}}},{Z_{56} = {V_{AC}\frac{I_{56}}{I_{5}I_{6}}}},{Z_{58} = {V_{AC}\frac{I_{58}}{I_{5}I_{8}}}}} & \left( {{Eq}.1} \right)\end{matrix}$

where V_(AC) is the amplitude of the applied AC voltage, I_(xy) is themagnitude of the AC current measured by electrode y when the AC signalis applied to electrode x, and I_(x) [I_(y)] is the sum of the magnitudeof the AC currents measured by all other electrodes when the AC signalis applied to electrode x [y]. The edge normalized impedance values arethen calculated as,

$\begin{matrix}{{Z_{51} = {\frac{V_{AC}}{\sqrt{2}}\frac{I_{51}}{I_{5}I_{1}}}},{Z_{53} = {\frac{V_{AC}}{\sqrt{2}}\frac{I_{53}}{I_{5}I_{3}}}},{Z_{57} = {\frac{V_{AC}}{\sqrt{2}}\frac{I_{57}}{I_{5}I_{7}}}},{Z_{59} = {\frac{V_{AC}}{\sqrt{2}}\frac{I_{59}}{I_{5}I_{9}}}}} & \left( {{Eq}.2} \right)\end{matrix}$

where the square root of 2 was determined to normalize the difference indistance between the edge and corner electrodes. The center normalizedimpedance value is then determined as,

Z ₅₅=median(Z ₅₂ , Z ₅₄ , Z ₅₆ , Z ₅₈)  (Eq. 3)

The use of the cross-electrode currents not only increases the effectivespatial resolution in comparison to using the max of the currentdistribution but it also allows for unadhered cells, which cause adecrease in the cross-electrode current, to be mapped.

FIGS. 5 a and 5 b illustrate an example of up-scaled cross-electrodeimpedance mapping in comparison with a fluorescent microscopy image.FIG. 5 a shows a fluorescent microscopy image 501 across the electrodearray and a heat map plot 502 of the normalized cross-electrodeimpedance of a cell culture immediately following plating. The enlargedmap 504 of a portion of heat map 502 shows a decrease in thecross-electrode normalized cell-substrate impedance Zs for the unadheredcells with single-cell resolution. The mapping immediately following aplating of cells such that the cells are not adhered shows smallernormalized impedance values where the cells are in comparison tonon-covered electrodes.

FIG. 5 b shows a fluorescent microscope image 505 and a cross-electrodeimpedance map 506 after 24 hours of culture. FIG. 5 b also shows anenlarged map 507 that is an overlay of fluorescent microscope image andcross-electrode impedance map at a select region. The results show thatmany of the cells have adhered to the surface, causing a drasticincrease in the normalized cross-electrode impedance.

Example 3: Quantifying Cell Adhesion

This example describes a method using cross-electrode impedance mappingto quantify cell adhesion.

Ethylenediaminetetraacetic acid (EDTA) is applied to the cells. EDTA isa calcium chelator that removes Ca²⁺ needed for integrin proteins tomaintain cell adhesion. With EDTA applied, the cells quickly detach overthe time course of ˜50 min. The EDTA is then washed out by adding normalculture media, where the cells re-attach over the time course of ˜200min.

The detachment and reattachment of the cells is captured with highspatial and time resolution using cross-electrode impedance mapping, asdemonstrated in FIG. 6 a , which shows a series of normalized impedancemaps over time of MDCK cells with a 5 mM EDTA application at t=˜5 minand a washout at t=˜55 min.

FIG. 6 b is a data plot showing mean normalized impedances for differentregions of the cell culture over time as specified in the map 601. FIG.6 cc are histograms of the normalized impedance values before, during,and after a washout of EDTA across the array.

To show a biologically relevant example of quantifying cell adhesion, agenetically modified MDCK cell line was measured wherein tetracyclinewas used to turn on and off a RasV12 and GFP gene. The result is shownin FIG. 7 . FIG. 7 is a series of fluorescent microscope images andnormalized cross-electrode impedance maps of MDCK cells over 7 days ofculture in vitro (DIV). Tetracycline is added after the 2 DIVmeasurement to turn on the gene RasV12 which is related to cancer, thegene also expresses GFP such that the gene expression can be imaged. Thetetracycline is then removed after the 4 DIV measurement to turn off thegene expression. The cells are shown to be less adherent to the surfacewhen the RasV12 gene is expressed and returns to normal after it isturned off.

RasV12 is an oncogene and has been known to increase cell metabolism anddecrease cell adhesion when strongly expressed, which together causecancer-like cell growth and tumors. Originally, tetracycline was keptout of the media and the cells were adhered as normal. When tetracyclinewas introduced, the genes were expressed causing an increase in GFP anda decrease in cell adhesion. Removal of tetracycline then reversed thecell adhesion to cause the cells to more strongly adhere while alsodecreasing overall GFP expression; some portions of the cell culture didnot turn off as strongly as others. The effects on cell adhesion werequantitatively compared to a control culture which did not havetetracycline introduced, as shown in FIG. 8 . FIG. 8 b is a normalizedimpedance histogram of MDCK cells over 6-7 days of culture in vitro(DIV). Tetracycline is added after the 2 DIV measurement to turn on thegene RasV12 which is related to cancer. FIG. 8 a is a normalizedimpedance histogram of a control measurement without the tetracyclineadded. The histograms have been normalized to the max pixel number abovethe no-cell impedance values of ˜8 kΩ. The cell adhesion was reduced incomparison to the control, which showed a smaller decreasing trend overtime.

Example 4: Frequency Response

This example describes the effect of frequency used in thecross-electrode impedance measurements.

The frequency of mapping was swept to determine the best frequency formeasuring the cell adhesion using cross-electrode impedance mapping.FIG. 9 shows a series of normalized cross-electrode impedance maps underdifferent frequency stimulus signals. The plots are normalized to themedian +/−1 standard deviation. The lower frequencies show higher signalcontrast which correlates to the optically measured GFP fluorescence asshown in FIG. 7 , which indicates that low frequency is better formeasuring cell adhesion. The used 1.9 kHz still shows good contrast incomparison to the 240 Hz, but above 10 kHz, the cell sheet looks muchmore uniform.

Example 5: Cell-to-Cell Adhesion

The previous examples are directed to how to map cells and theiradhesion over time via a cross-electrode impedance measurement, asdepicted in FIG. 10 a . In FIG. 10 a , an AC voltage is applied to asingle electrode and the currents are measured through the remainder ofthe electrode array using transimpedance amplifiers. The adhesion ismainly a function of the cell-to-substrate attachment and resultantheight of the gap.

This example describes a method to measure cell-to-cell attachment, orhow well connected the cells are to each other. Cells in culture notonly attach to the surface, but also to each other via cell-cellconnections. The tightness of these connections defines the permeabilityof a cell sheet and is important for epithelial tissues which act asbarriers of the body surfaces, internal organ linings, and othertissues. In this example, this barrier function is measured byperforming a map of the transepithelial impedance, Z_(te). In this way,the cell-cell connectivity could be assessed using electrodes onlycovered by cells to mitigate any holes while also allowing for spatialheterogeneity assessment.

In this example, the stimulation protocol is modified to measure thevertical field component 1014 as shown in the diagram in FIG. 10 b . InFIG. 10 b , an electrode 1006_2 and its surrounding electrodes 1006_1,1006_3 are biased with an AC voltage. A current Ie,n is measured throughthe center electrode 1006_2. The center electrode 1006_2 will not passcurrent to surrounding electrodes as they are biased with the samesignal, therefore it will only pass current due to the impedance of cellsheet above the electrode. Outside of the center and its surroundingelectrodes, the remainder of the array is biased at ground or areference voltage level to act as a current return. This type ofmeasurement is similar to measuring the transepithelial electricalresistance (TEER), which is measured using two electrodes on oppositesides of a cell culture on a suspended porous membrane. The techniqueshown in FIG. 10 b allows the TEER to be mapped across the cells on topof the electrode array without the need for special suspension.Advantages include fewer cells needed, ability to assess spatialheterogeneity, and the ability to combine cell-to-cell andcell-to-substrate adhesion measurement using the same device.

FIG. 24 includes schematic diagrams illustrating some additional schemesof cell-cell connectivity measurements, in accordance with someembodiments. In FIG. 24 , the change of the vertical field above theelectrode is measured to best isolate the effects of the cell-cellconnections using two circuit configurations: 1) a fast (<1s/measurement) parallel electrode measurement versus a reference (FIG.24 a ), and 2) a slow scanned (40 s/measurement) relative measurementwithout a reference (FIG. 24 b ). The fast measurement is ideal forsweeps across multiple frequencies whereas the scanned measurement doesnot require a reference which helps to make long-term measurements morestable and is more ideal for device miniaturization. For both types ofmeasurements, platinum black (PtB) deposition can be optionally used tolower Z_(e) by about 5× to improve Z_(te) sensitivity. Experimentsacross frequency showed that mid-range frequencies of ˜2 kHz to 5 kHzwere best for assessing cell-cell connectivity.

The calculation of transepithelial impedance Zte using the schemes inFIG. 24 is now discussed below.

To measure cell-to-cell attachment, or how well connected the cells areto each other, we can modify the stimulation protocol to measure thevertical field component in FIGS. 24 a, 24 b . Measurements can be madeversus a grounded reference (left) by applying an AC voltage to allelectrodes with the each transepithelial electrode current, I_(te,n)(n=1, 2, . . . 4096), measured via transimpedance amplifiers(measurement duration of 1 s/frequency). The resultant fielddistribution is vertically aligned with the connectivity of the cellsdecreasing the I_(te). A non-reference measurement can be made (right)by applying an AC voltage to an electrode (n) and its neighboringelectrodes to create an effective vertical field measurement with theremainder of the electrodes' grounded. To generate a cell map, theapplied signal is scanned across the array (40 s per scan/frequency).

In the parallel scheme in FIG. 24 a , an AC voltage is applied to eachelectrode versus a reference with each electrode's current, I_(te,n),measured, creating a vertical field in solution (the peripheralelectrodes would also have a fringing field for low frequencies. Due tocurrent then needing to go through the cell sheet, the magnitude of thecurrent will be proportional to the transepithelial impedance, Z_(te). Asecond scanned scheme, FIG. 24 b , biases an electrode and itssurrounding electrodes with an AC voltage and measures the current onlythrough the center electrode. The center electrode will not pass currentto surrounding electrodes as they are biased with the same signal,therefore it will only pass current due to the impedance of cell sheetabove the electrode. Outside of the center and its surroundingelectrodes, the remainder of the array is biased at ground to act as acurrent return.

In either case, the measured vertical current I_(te,n) can be expressed,

$\begin{matrix}{I_{{te},n} = \frac{V_{A}}{Z_{e,n} + Z_{te}}} & \left( {{{Eq}.A}8} \right)\end{matrix}$

Using (A3), we can then solve for Z_(te),

$\begin{matrix}{Z_{te} = {\frac{V_{A}}{I_{{te},n}} - \frac{V_{A}}{I_{n}}}} & \left( {{{Eq}.A}9} \right)\end{matrix}$

For measurements, it was determined that mid-frequencies from ˜1-5 kHzare best correlated with the cell-cell connectivity (see also Example15, below). For the PtB electrodes, Z_(e,n) is then sufficiently smallerthan Z_(te) (see also Example 15, below) such that it is estimated that:

$\begin{matrix}{Z_{te} = \frac{V_{A}}{I_{{te},n}}} & \left( {{{Eq}.A}10} \right)\end{matrix}$

For Z_(te) experiments with just Pt electrodes, the I_(n) measurementfrom the cell-substrate impedance is subtracted. Due to the scannedarray measurement to calculate Z_(te,no ref) using a 3×3 set ofelectrodes, the total map generated is 62×62 pixels, as the peripheralelectrodes do not have neighboring biased electrodes to create thevertical field. The measurement versus the reference creates a mapcontaining 64×64 pixels.

Example 5A: Metabolic State Mapping Via Extracellular Redox Potential,Vredox

Beyond impedance measurements, platinum electrodes have been used forboth potentiometric sensing of oxygen and extracellular redoxmonitoring. This example demonstrates that we could use the proximatelocation of Pt electrodes directly underneath live cells to map theextracellular redox potential, V_(redox), in situ to monitor the redoxenvironment of the cells and even O₂ consumption to map out themetabolic state of cell cultures.

To accomplish the measurement, the pixel amplifier is configured as abuffer, as shown in the schematic diagram in FIG. 25 a.

In general, cells use energy arising from the movement of electrons fromoxidizable organic molecules (e.g. glucose) to O₂ during aerobicmetabolism. To help mediate these electron flows, a general reducingenvironment is created by the thiol-compound glutathione (GSH) which isoften considered to be the cellular redox buffer. In simplified terms,the redox potential of the cell is then a balance between O₂ pulling thepotential up (oxidizing) and GSH pulling the potential down (reducing).The redox environment is not only important for electron transfer, butalso for neutralizing harmful reactive oxygen species, cell-cellsignaling, and regulating the state of the cell. For example, rangingfrom negative to positive, the redox potential can determine if a cellis in a state of proliferation, differentiation, apoptosis, or necrosis.

FIG. 25 b are a series of data maps showing results of multi-parametricmeasurements. The measurements are performed at +24, +48, and +72 hoursafter MDCK cell plating including cell attachment (top), cell-celladhesion (middle), and metabolic state (bottom). The cells exhibitgrowth from the bottom right to the upper left corner, where theproliferating leading edge cells proliferating show the most negativeV_(redox) in comparison to the more dormant trailing edge. The Z_(te) ishighest at the leading edge as well, due to the lowest density of cells,see detail region 1, and therefore the fewest cell-cell connections.FIG. 25 c is a pair of nuclei fluorescence images at +72 hours afterplating (top) and a detail region 1 comparison (bottom) showing thelowest cell density on the leading edge in comparison to the trailingedge. FIG. 25 d is a composite map showing a detail region 2 overlayingthe cell nuclei and cell attachment. FIG. 25 d shows good spatialcorrespondence with single-cell resolution.

One goal of this example is to investigate what information theproximate V_(redox) could provide by pairing it with the impedancetechniques to monitor cell growth (FIG. 25 b ). In this example, anegative V_(redox) in the range of 30 mV to 80 mV was observed forelectrodes with cells in comparison to electrodes without cells (FIG. 25b ). From the detail region comparison, the spatial information ofV_(redox) is distinct and different than the cell attachment or cellbarrier, where the most negative V_(redox) is at the leading edge andnot the lowest density. In general terms, the negative signal couldindicate a locally smaller [O₂] or locally higher [GSH] near the cells.

To further explore the Vredox signal origin, the O₂ dependence wastested via an oxygen purge on a separate MDCK cell culture. Upon the O₂removal, the signal difference between regions with cells and withoutwas eliminated. To complement, the GSH based reducing capacity wastested via an oxidative titration. Ferricyanide, [Fe(CN)6]³⁻, was chosenfor the titration due to its previous non-toxic use in cell-cultures andits oxidizing half-cell potential in comparison to the cellularenvironment. The media showed a 4 μM reducing capacity while the cellshad a much larger capacity of >200 μM.

Taken together, these measurements show that the measured V_(redox) isrelated to both the in situ [O₂] and [GSH]-based reducing capacity ofthe cells. We theorize that with aerobic respiration, the [O₂] lowersfrom its normal dissolved concentration of ˜200 μM at atmosphericconditions which lowers V_(redox) until it is regulated by theextracellular reducing potential of the cells. Therefore, though it isdifficult to quantify oxygen consumption rate with our technique, theV_(redox) measurement of the extracellular redox potential can be usefulfor monitoring the metabolic state of cells, as it can show both theusage of O₂ and the reducing environment of cells. Therefore, the morenegative signal on the leading edge of the cell sheet (FIG. 25 b ) isattributed to respiration combined with a state of proliferation, themost negative redox potential state of a cell.

Example 6: Antibody-Cell Binding

Screening for antibody-cell binding can be low-throughput due the needfor either fluorescent tagging of the antibodies, which requires washsteps to remove non-bound fluorescent antibodies, or the need for aspecial optical measurement such as surface plasmon resonance (SPR).According to one aspect, the cross-electrode impedance techniquedescribed herein may offer the ability to measure the antibody-cellbinding event through either the cell-to-substrate or cell-to-celladhesion measurements. With the binding of an antibody on the undersideof the cell, the gap distance becomes effectively smaller leading to adecrease in the amount of cross-electrode current measured. Likewise,with antibody binding to the sides of the cells, the cell-to-cell gapdistance should also become smaller, leading to a decrease in the amountof vertical current measured. Being able to perform such antibodybinding without labels then allows for different antibodies to be addedin sequence without the need for wash steps, greatly improvingthroughput.

Example 7: Cell Patterning Through Electrochemical Gas Generation

This example describes a method to pattern cells on top of an electrodearray. The inventors have recognized and appreciated that small gasbubbles can be electrochemically generated to generate small holes inthe cell membrane to kill the cells via depolarization. After death,cells will then detach from the surface, as illustrated in the schematicdiagram in FIG. 11 . Therefore, by controlling which electrodes generategas, the cells can be patterned with the spatial resolution of theelectrode array.

Without wishing to be bound to a particular theory, the inventorsrecognized that for most inert electrode materials (platinum, gold,etc.) hydrogen gas can be generated by adjusting the electrode potentialbelow the hydrogen ion/hydrogen gas redox half-cell reduction potential)(E⁰),

2H⁺+2e ⁻

H₂(g)E°=0.00V  (Eq. 4)

or oxygen gas can be generated by adjusting the electrode potentialabove the oxygen gas/water redox potential,

O₂(g)+4H⁺+4e ⁻

2H₂O E⁰=+1.23V  (Eq. 5)

Likewise, as most cell media contain chloride salts, chloride gas mayalso be generated by adjusting the electrode potential above thechlorine gas/chloride redox potential,

Cl₂(g)+2e ⁻

2Cl⁻E°=+1.36V  (Eq. 6)

Accordingly, cell removal may be performed by selectively applying apre-determined potential that is above a redox potential for generationof a gas at one or more electrode locations. The potential may beapplied, for example by connecting one or more stimulus source circuits110 in FIG. 1 a to the selected electrodes. The potential needs not tobe identical across all selected electrodes, and programmableheterogeneity may be used when electrodes are biased differently. Thepotential may be a potential relative to a potential of a referenceelectrode in the medium above the electrodes.

For more controllable patterning, an electrode current can be used toset the electron transfer rate and therefore the rate of gas generation.Controlling the rate of gas generation can optimize the selectiveelectrochemical reaction as large bubbles can form on the surface byusing too fast of a gas generation rate, blocking the electrodes fromsolution.

FIG. 13 is a series of diagrams illustrating variations of cellpatterning using an electrode array. FIGS. 13 a and 13 b illustrateembodiments where one or more pre-determined patterning voltages areapplied to selected electrodes for patterned removal of a cell byelectrochemical gas generation. FIGS. 13 c and 13 d illustrateembodiments where one or more pre-determined patterning currents areapplied to selected electrodes for patterned removal of a cell. FIGS. 13a and 13 c illustrate an example of voltage/current patterning with areference electrode acting as a return. FIGS. 13 b and 13 d illustratean example of differential voltage/current patterning usingcross-electrode gas generation without using a reference electrode,where one set of electrodes passes a positive current and a second setof electrodes passes a negative current (return).

Example 8: Cell Spatial Patterning and Defining a Co-Culture

This example describes spatial patterning of cells and definition of aco-culture using an electrode array.

A CMOS electrode array as shown in FIG. 12 , MDCK cells, and H₂ gasgeneration are used in this example. In this experiment, H₂ gas wasgenerated by applying −1.25 V to the platinum electrodes versus aAg/AgCl pseudo reference electrode. FIG. 12 shows fluorescent microscopeimages of before (middle) and after (right) patterning voltage isapplied for 80 seconds, and show that the pattern in cells was definedsuccessfully based on the pattern of electrodes. With the electrodepitch of 20 μm, square holes of various sizes were made in the uniformcell sheet with high spatial resolution, as confirmed using nucleifluorescent markers and fluorescent imaging.

FIG. 14 shows a series of fluorescent microscope images illustrating theprocess of defining a co-culture via patterning and then plating asecond cell type. The cell types were distinguished by different nucleifluorescent markers. In the experiment in FIG. 14 , a co-culture of twodifferent cell types was defined by plating a second MDCK cell line witha different nuclei fluorescent marker after the initial patterning. Thesecond cell type filled in the generated space, showing the ability tospatially define co-cultures with high spatial resolution. Furtherpatterning and plating could also be performed to define multiple cellco-cultures and patterns in a bottom-up approach.

Example 9: Directed Cell Evolution by Removing Culture Heterogeneity

This example describes a method of directed cell evolution to eliminatecells from the cell culture whose properties are not desired.

FIG. 15 is a series of schematic diagrams illustrating a heterogeneouscell population, elimination of undesired cells using patternedelectrochemical gas generation on select electrodes, and a homogenousculture of desired properties after subsequent cell growth. The choiceof which cells to eliminate can be made via optical imaging or via otherproperties measured using the electrode array. The capability toeliminate cells from the culture without having to remove from theculture plate is advantageous over current processes which would requiresuspending the cells and separating using a cell sorting machine with afurther replating step to again culture, or removal of single cells withthe desired properties using a micropipette and then replating.Furthermore, the lineage of the cell history can be preserved as thespatial location of each cell does not change as the cells remainadhered during the process. Such an elimination process could also beused for further analyses to be performed to on a subset of cells afterculturing the electrode array, where cells unwanted for furthermeasurement are first killed before cell suspension and removal.

Example 10: Wound Healing Assay

This example describes a combined application of both thecross-electrode impedance mapping and cell patterning is a wound healingassay.

These assays gauge cell growth rate and metabolism and can be useful forscreening drugs affecting these parameters. Compared to theelectrochemical patterning described herein, other tools mechanicallygenerate a wound in a cell culture via a mechanical scratch which isboth difficult to control and limiting in terms of wound pattern.

In this example, a wound is made in MDCK cells and then the growth ismapped in real-time. FIG. 16 shows a sawtooth-like pattern in the cellsdefined in the center of the device surface with varying distances ofseparation. These patterns were defined by applying electrode currentsof −10 nA for 40 s versus a Ag/AgCl pseudo reference electrode. Theregrowth of the culture was then measured using the impedance mappingmethod. A typical cell culture took ˜3 days to fill in the wound whereasa culture with a growth inhibition drug showed very little regrowth. Asillustrated by the normalized cross-electrode impedance maps in FIG. 16, the control culture shows regrowth after 72 hours in culture. A secondculture with a drug that slows growth, cytochalasin B (1 μM), shows verylittle growth over the course of 72 hours, demonstrating the ability ofthe assay for drug screening.

Example 11: Molecular Delivery

This example describes a technique using planar electrodes for membranepermeabilization and molecular delivery. Unlike electroporation, whichapplies a concentrated electric field to break down the cell membrane,planar electrode permeabilization works via gas bubble formation,similar in concept to the patterning techniques discussed herein. Unlikepatterning cells, where cells are killed to perform the patterning, formolecular delivery smaller holes are created on the cells that will thenreseal over time.

FIG. 17 illustrates an experiment demonstrating permeabilizationtechniques using nanowire electrodes, while aspects of the technique mayalso be applicable using an electrode array using planar electrodes. Inthe experiment shown in FIG. 17 , Fluo-4, a live assay, is dissolved inthe extracellular solution (left panel, FIG. 17 a ). Electroporationprotocols are applied to the nanoelectrodes using the pixel stimulator(middle panel, FIG. 17 a ) and allowed to recover in the Fluo-4. Ifsuccessfully electroporated, Fluo-4 permeates into the cell. Afterrecovery, a dead assay, EthD-1 is dissolved in the extracellularsolution to reveal if cells have died due to irreversibleelectroporation (right panel, FIG. 17 a ). Cells that are successfullyelectroporated and recover retain Fluo-4 for imaging. FIG. 17 b shows aheat maps showing the EthD-1 and Fluo-4 intensity averaged across eightinvestigated protocols of increasing voltage amplitude (3 trains of 5biphasic pulses at 20 Hz) performed with HEK 293 cells. The CNEA arrayis divided into subgroups of 128 pixels for each of the eight protocolsand repeated in a grid across the array. Imaging is performed on eachpixel and the 128 images for each protocol are averaged together. FIG.17 c shows the averaged intensity results from FIG. 17 b for the HEK 293cells. Successful electroporation is viewed starting at ˜1.3 V, whereasirreversible electroporation starts ˜1.7 V. FIG. 17 d shows results withneurons for the same test conditions show a lower threshold forsuccessful electroporation, <1.2 V, and irreversible electroporation˜1.5 V.

FIG. 18 illustrates another experiment, in which Fluo-4 is injected intothe cell using Fluo-4 AM. Electroporation protocols are applied to thenanoelectrodes using the pixel stimulator (middle) while thefluorescence is monitored. If successfully electroporated, Fluo-4 isable to flow out of the cell causing a decrease in fluorescence. Forsuccessful protocols, the cell membrane recovers after electroporation(right panel, FIG. 18 a ). FIG. 18 b illustrates an example of using aneuron with its fluorescence and applied electroporation signal. Duringelectroporation, the fluorescence drops. Immediately afterwards, thecell membrane recovers and causes the fluorescence to plateau. Theelectroporation signals may be applied multiple times without affectingcell viability.

In both experiments shown in FIG. 17 and FIG. 18 , it was observed thatvoltage signals needed to be of a certain duration, at least >50 ms, tosee any permeabilization/delivery. This points to the need for Faradaicprocesses to generate gas bubbles, as the voltage needed is alsocomparable to the water window voltages (H₂ & O₂ gas generation viawater splitting) with the platinum electrodes used. In FIG. 18 , such apermeabilization signal is shown to be effective by causing transientleakage of a fluorescent dye, while in FIG. 17 , a fluorescent dye isdelivered to the cells.

Such delivery capabilities can be readily used for screening membraneimpermeable compounds for their effects on cells and cell-to-cellinteractions. The spatial capabilities of the electrode array, in whichcells can be chosen for delivery, can be useful in this latterapplication of cell-to-cell interactions where the delivered cell andits undelivered neighbors can be measured for the effects of thecompounds. Without such delivery capabilities, the membrane impermeablecompounds would otherwise need to be chemically modified for delivery,which is expensive and time-consuming, or delivered using a micropipetteon a single-cell basis, which is also expensive and time-consuming.Beyond compounds, RNA/DNA/plasmids can also be delivered forapplications to synthetic biology.

Example 12: Serial Delivery for Cross-Effect Analysis

This example describes a multi-step delivery of compounds in cells usingan electrode array.

FIG. 19 shows a series of schematic diagrams illustrating generation ofa control and cross-effect delivery using spatial addressing and serialdelivery via gas generation. As the electrode's properties are notmodified during the gas evolution, in combination with the spatialcapabilities of the addressable electrodes offers a further advantage ofcross-compound effect screening. For example, if two compounds aredesired to be investigated for their effects on cells, just two compounddelivery steps are needed to form a complete matrix of drug effects.

Example 13: Extracellular Electrochemical Mapping

This example describes electrochemical mapping using redoxelectrochemistry on the electrode array.

Electrochemical measurements of cells using electrodes can use a single,large working electrode to measure bulk concentrations of analytes insolution. Such electrochemical electrode-based measurements include theClark electrode for dissolved oxygen concentration measurement andhydrogen ion concentration (pH) measurement. According to an aspect ofthe present disclosure, an array of electrochemical electrodes may beused to spatially map analyte concentrations measured via electronicswithin a CMOS integrated circuit. Such electrochemical mapping can thenbe applied for cell analysis of cells cultured directly on top of theelectrode array.

In this example, to demonstrate the capability for electrochemicalmapping using an array of electrodes measured using a CMOS integratedcircuit, cyclic voltammetry is performed using a common redox couple offerricyanide/ferrocyanide, [Fe(CN)₆]³⁻/[Fe(CN)₆]⁴⁻.

[Fe(CN)₆]³⁻+1e ⁻

[Fe(CN)₆]⁴⁻E°=+0.36V  (Eq. 7)

FIG. 20 a is a schematic diagram showing the cyclic voltammetryconfiguration using CMOS integrated transimpedance amplifiers to measureeach Pt electrode's current and an external transimpedance amplifier tomeasure the current through a Ag/AgCl pseudo reference electrode. In theexperiment in FIG. 20 a , a cyclic voltage ramp was applied at a scanrate of 35 mV/s with 1.5 M KCl+5 mM K3[Fe(CN)6]. The sum of the 13×13electrodes' currents is used for the measurement, which equals that ofthe reference electrode. FIG. 20 b show two spatial maps of the maxrange of the electrodes' currents (|_(Ie,max)−I_(e,min)|; top left)which is related to the diffusion of ferricyanide (starting reactant),and the max range of currents minus the max/min voltage currents(|I_(e,max)−I_(e,min)|−|I_(e,vmax)−I_(e,vmin)|; bottom left) which isrelated to the ferrocyanide diffusion (product). Example individualelectrode recordings are shown in FIG. 20 b on the right with theseparameters defined. The non-radial ferrocyanide diffusion is attributedto convection effects in solution.

In this experiment, a subset 13×13=169 of a 64×64 array of electrodeswas connected to the same number of respective transimpedance amplifierswith a cyclic linear voltage ramp applied, as illustrated in theschematic diagram in FIG. 20 a . FIG. 20 b show spatial maps of thecurrent density that show increased cathodic and anodic currentmagnitudes on the edges of the electrode, which may be attributed toincreased radial diffusion/mass transport of the edge in comparison tothe planar diffusion of center electrodes. Likewise, generation ofproducts limits current density which is visualized by the peak currentrange minus the voltage maximum/minimum current range, as illustrated inthe data plot 2001 in FIG. 20 . The cyclic voltammetry data plot 2001shows a tendency for product diffusion towards the upper right corner.The spatial measurement of such current shows the capability forcurrent-based electrochemical mapping.

The open-circuit potential of the electrodes can also be used to measurethe concentration of chemical species in solution. For ahigh-concentration of a redox couple in solution, the open-circuitpotential of platinum electrodes in solution can be determined by theNernst equation. The Nernst equation relates the reduction potential ofan electrochemical reaction to the standard electrode potential,temperature, and activities of the chemical species undergoing reductionand oxidation,

$\begin{matrix}{\left. {{Ox} + {ne}^{-}}\rightleftharpoons{Red} \right.{E_{H} = {E^{0} - {\frac{\varphi_{t}}{n}\ln\frac{\lbrack{Red}\rbrack}{\lbrack{Ox}\rbrack}}}}} & \left( {{Eq}.8} \right)\end{matrix}$

where E_(H) is the electrode voltage potential with respect to thestandard hydrogen electrode (S.H.E), E⁰ is the half-cell reductionpotential, φ_(t) is the thermal voltage (˜25.7 mV at 25° C.), [Ox]/[Red]is the concentration of the oxidized/reduced chemical species, and n isthe number of electrons transferred in the cell half reaction. For theferricyanide/ferrocyanide reaction, measurement of the open-circuitpotential then reflects the ratio of the concentrations of these ions insolution.

In this example, the potential of the remainder of the electrode arraywas measured. In particular, ferrocyanide generation and transportacross a CMOS electrode are mapped using open circuit potential.

A cyclic potential is applied to a group of 13×13 electrodes (with 9electrodes excluded within the group of 13×13 electrodes, as illustratedin FIG. 21 b ) while the remaining electrodes' open circuit potentialsare measured. FIG. 21 a are data plots that show select electrodevoltages, V_(el), plotted over time, which shows an increase anddecrease related to the ferricyanide/ferrocyanide concentrations. FIG.21 b is a heat map that illustrates for one cycle, the overall amplitude(maximum minus the minimum) of the open circuit potential plotted acrossthe array to show diffusion/mass transport which tends towards the upperleft corner. FIG. 21 c shows a heat map and a data plot that illustratethe minimum time of the open-circuit potential plotted versus distancefrom the center of the 13×13 electrodes showing the transient aspects ofthe diffusion/mass transport.

In summary, for the cyclic voltammetry, measuring the open-circuitaround the electrode shows the flow of ferrocyanide towards the upperright corner of the device.

Example 14: Electrochemical Oxygen Mapping of Cells

This example describes a technique applying electrochemical mapping tocell analysis. For example, a Clark electrode based on platinum may bemeasured by applying a pulsed voltage or a voltage pulse sequence whichsequentially oxidizes and then reduces the platinum. As platinum oxideblocks oxygen reduction, the current drops to zero after the oxide isformed. When the oxide is then reduced, the platinum electrode passes anegative current due to the presence of oxygen,

O₂(g)+4H⁺+4e ⁻

2H₂O E⁰=+1.23V  (Eq. 9)

The local oxygen concentration is then consumed, and the electrode waitsfor the further diffusion of additional oxygen to the electrode to passcurrent. Therefore, the rate of the equation is limited by oxygendiffusion which is proportional to the oxygen concentration in solutionand can be measured by measuring the electrode current.

In an experiment using an electrode array, a measurement was performedusing a salt solution (phosphate-buffered saline) exposed to ambient airand then subsequently purged with nitrogen gas to reduce the oxygenconcentration. FIG. 22 a shows a voltage pulse sequence 2202 applied tostimulation electrodes in the electrode array, and a series of dataplots 2204 of measurement using the CMOS electrode array in ambient air,with a partial nitrogen purge, and a partial recovery (N₂ purgestopped), respectively. The data plots 2204 show that the electrodecurrent reflects the oxygen concentration.

Comparing the current I_(el) before and after the purging shows a markedreduction. Experiments were then performed with HEK293 cells, and theresults are shown in FIG. 22 b . FIG. 22 b shows a cross-electrodeimpedance heat map 2206 using cross-electrode max current I_(max) overthe electrode array area, and a heat map 2208 of a change in electrodecurrent ΔI_(el) over the electrode array area. The same style of oxygenmeasurement with HEK293 cells shows a decrease in oxygen concentrationwhere the cells are located, as confirmed with an impedance map.

Cells consume oxygen as a part of aerobic metabolism, therefore theoxygen concentration around cells is smaller than places without cells.Indeed, mapping the electrode current across the array shows thelocation of the cells has a smaller magnitude of current than placeswithout cells, as imaged using a cross-electrode impedance map. The leftand bottom edges of map 2208 also show a larger magnitude of current,which is attributed to edge effects and the increased diffusion/masstransport.

Example 15: Effects of Platinum Black and Frequency on Cell BarrierSensitivity

In this example, platinum black (PtB) was used to lower the electrodeimpedance, Z_(e), to improve cell barrier measurement sensitivity. FIG.26 a shows results of a comparison study of electrode impedance forcells cultured at ˜72 hours with electrodes under three scenarios:low-density, high-density, and without cells. FIG. 26 b is a data plotillustrating that PtB lowered the Z_(te) measurement of bare electrodesby about 5×, allowing the cell-cell connections of the two differentdensities to be measured with higher signal-to-noise. FIG. 26 cillustrates cell barrier maps versus a reference at differentfrequencies. The lower frequency measurements show more spread and donot capture the cell sheet edge but the 1.8 kHz measurement showed thehighest contrast for the cell-connection measurement when compared todensity maps extracted from imaging. FIG. 26 d shows cell density andconnectivity maps extracted from the nuclei of the fluorescence images.FIG. 26 e shows a comparison between Z_(te) measured without and with areference at 1.8 kHz. Slightly smaller Z_(te) is measured without thereference, but for regions with cells and without (the two clusters) therelationship is direct. Measurements without the reference arepreferred, as the Z_(e) contribution can be easily subtracted from thecell-substrate attachment measurement. FIG. 26 f shows a comparisonbetween Z_(te) and Z_(s) versus extracted cell density. For thiscomparison, Z_(s) is down-sampled via a bilinear interpolation to havethe same spatial resolution as the Z_(te) measurement. The cell barriershows a stronger dependence on cell density due to its measurementgeared towards cell-cell connectivity. There's a small correlationbetween Zs and cell density as well, which can be seen from thecell-circuit model (FIG. 4B) as having an effect if Z_(s) is high andthe assumption that Z_(s)<<Z_(te) no long holds which was used in theZ_(s) calculation.

Having thus described several aspects of at least one embodiment of thisinvention and examples thereof, it is to be appreciated that variousalterations, modifications, and improvements will readily occur to thoseskilled in the art. Such alterations, modifications, and improvementsare intended to be part of this disclosure, and are intended to bewithin the spirit and scope of the invention. Further, though advantagesof the present invention are indicated, it should be appreciated thatnot every embodiment of the technology described herein will includeevery described advantage. Some embodiments may not implement anyfeatures described as advantageous herein and in some instances one ormore of the described features may be implemented to achieve furtherembodiments. Accordingly, the foregoing description and drawings are byway of example only.

Various aspects of the present invention may be used alone, incombination, or in a variety of arrangements not specifically discussedin the embodiments described in the foregoing and is therefore notlimited in its application to the details and arrangement of componentsset forth in the foregoing description or illustrated in the drawings.For example, aspects described in one embodiment may be combined in anymanner with aspects described in other embodiments.

Also, the invention may be embodied as a method, of which an example hasbeen provided. The acts performed as part of the method may be orderedin any suitable way. Accordingly, embodiments may be constructed inwhich acts are performed in an order different than illustrated, whichmay include performing some acts simultaneously, even though shown assequential acts in illustrative embodiments.

Use of ordinal terms such as “first,” “second,” “third,” etc., in theclaims to modify a claim element does not by itself connote anypriority, precedence, or order of one claim element over another or thetemporal order in which acts of a method are performed, but are usedmerely as labels to distinguish one claim element having a certain namefrom another element having a same name (but for use of the ordinalterm) to distinguish the claim elements.

The terms “approximately” and “about” may be used to mean within ±20% ofa target value in some embodiments, within ±10% of a target value insome embodiments, within ±5% of a target value in some embodiments, andyet within ±2% of a target value in some embodiments. The terms“approximately” and “about” may include the target value.

1-22. (canceled)
 23. A method for providing spatially positionedelectrochemical reactions with an electrode array exposed on a surfaceof a semiconductor substrate, the electrode array exposed to a solutionincluding one or more chemical or biological reagents and a plurality ofcells attached to the semiconductor substrate, the method comprising:selecting one or more electrodes in the electrode array; controllingcircuitry in the semiconductor substrate to apply, at the one or moreelectrodes, one or more stimulus signals including at least one of apatterning voltage or a patterning current for generation of gas in thesolution to initiate an electrochemical reaction at the selected one ormore electrodes; and generating the gas at the selected one or moreelectrodes to cause at least one of permeabilization or detachment fromthe surface of the semiconductor substrate of at least one cell of theplurality of cells disposed on the selected one or more electrodes. 24.The method of claim 23, wherein the patterning voltage exceeds a redoxpotential for generation of gas in the solution.
 25. The method of claim23, wherein the permeabilization of at least one cell of the pluralityof cells comprises forming in the at least one cell holes that aresmaller than those formed when the at least one cell is detached fromthe surface of the semiconductor substrate.
 26. The method of claim 23,wherein the gas comprises H₂, Cl₂, or O₂.
 27. The method of claim 23,wherein the plurality of cells are a plurality of cells of a type, andthe method further comprises: plating one or more cells of a second typeon the surface of the semiconductor substrate at locations where the atleast one cell of the first type has detached from.
 28. The method ofclaim 23, further comprising: mapping a time sequence of regrowth of theplurality of cells on the surface at positions where the at least onecell has detached from; and based on the mapping, determining a growthrate of the plurality of cells.
 29. The method of claim 28, furthercomprising: measuring with the controlling circuitry an open-circuitpotential at each of some or all electrodes in the electrode array tomeasure an extracellular redox potential; and based on the mapping,determine a cellular metabolic state and respiration of the plurality ofcells during growth.
 30. The method of claim 28, further comprising:measuring with the control circuitry a current at each of some or allremaining electrodes in the electrode array.
 31. The method of claim 23,wherein the one or more stimulus signals are relative to a current or apotential of a reference electrode.
 32. The method of claim 23, whereinthe semiconductor substrate includes a plurality of voltage buffercircuits coupled with the electrode array, the method furthercomprising: selecting a set of electrodes in the electrode array; andcontrolling the voltage buffer circuits to measure an extracellularredox potential at each electrode of the set of electrodes.
 33. A systemcomprising: a semiconductor substrate comprising: an electrode arrayincluding a plurality of individually addressable electrodes disposed ona surface of the semiconductor substrate; a solution including one ormore chemical or biological reagents and a plurality of cells attachedto the semiconductor substrate; and circuitry that is controllable byone or more processors to apply, at a group of electrodes in theelectrode array, one or more stimulus signals including at least one ofa patterning voltage or a patterning current, relative to a potential ofor current at an electrode in the electrode array or a potential of orcurrent at a reference electrode, for generation of gas in the solutionto initiate an electrochemical reaction at the group of electrodes andgenerate gas at the group of electrodes to cause at least one ofpermeabilization or detachment from the surface of the semiconductorsubstrate of at least one cell of the plurality of cells disposed on thegroup of electrodes.
 34. The system of claim 33, wherein the electrodearray comprises a plurality of pads disposed on an insulative surface ofthe semiconductor substrate.
 35. The system of claim 34, wherein theplurality of pads comprises Au or Pt.
 36. The system of claim 33,wherein the reference electrode is a Ag/AgCl reference electrode. 37.The system of claim 33, wherein the electrode array comprises at least1000, at least 4000, or at least 1,000,000 electrodes, and the circuitrycomprises a plurality of recording circuits, each recording circuitconfigured to measure a current at an electrode of the electrode array.38. The system of claim 37, wherein the plurality of recording circuitscomprises at least 10 recording circuits, or at least 4000 recordingcircuits.
 39. The system of claim 37, wherein each recording circuitcomprises a transimpedance amplifier (TIA).
 40. The system of claim 39,wherein the TIA comprises an impedance component having a resistance ofat least 10 MOhm, wherein an output voltage of the TIA is proportionalto a voltage across the impedance component.
 41. The system of claim 40,wherein the impedance component comprises a switching capacitor.
 42. Thesystem of claim 33, wherein the circuitry further includes a pluralityof buffer circuits coupled with the electrode array, wherein thecircuitry is controllable by one or more processors to measure, via theplurality of buffer circuits, an extracellular redox potential at eachcell of a set of electrodes of the electrode array.
 43. A system forproviding spatially positioned electrochemical reactions, the systemcomprising: an electrode array exposed at a surface area of asemiconductor substrate; a solution including one or more chemical orbiological reagents and a plurality of cells attached to the surfacearea of the semiconductor substrate; circuitry disposed in thesemiconductor substrate and coupled to the electrode array; at least onenon-transitory computer-readable medium having stored thereon executableinstructions; and at least one processor programmed by the executableinstructions to perform a method comprising acts of: selecting a patternof electrodes in the electrode array; and controlling circuitry toapply, at the pattern of electrodes, one or more pre-determined stimulussignals including at least one of a patterning voltage or a patterningcurrent, relative to a potential of or current at an electrode in theelectrode array or a potential of or current at a reference electrode,for generation of gas in the solution such that gas is generated at thepattern of electrodes to cause at least one of permeabilization ordetachment from the surface of the semiconductor substrate of at leastone cell of the plurality of cells that is disposed on the pattern ofelectrodes.